A carfentanil molecular vaccine based on polylactic acid-polyethyleneimine and a preparation method and application thereof

Abstract

The application discloses a carfentanyl molecular vaccine based on polylactic acid-polyethylene imine and a preparation method and application thereof, and relates to a carfentanyl molecular vaccine MECV PLA‑PEI The carfentanyl molecular vaccine can produce a strong hapten-specific antibody response, can effectively avoid the generation of non-specific anti-carrier immunity, and can inhibit carfentanyl-induced analgesia and addictive behavior.

Description

Technical Field

[0001] This invention belongs to the field of vaccine development technology, specifically relating to a carfentanil molecular vaccine based on polylactic acid-polyethyleneimine, its preparation method, and its application. Background Technology

[0002] Fentanyl and its derivatives are a class of potent synthetic opioids. Due to their high lipid solubility and small molecular weight, they can easily cross the blood-brain barrier and rapidly enter the central nervous system. Because of their high affinity for μ-opioid receptors in the central nervous system, they can produce significant analgesic effects, with potency tens to thousands of times that of morphine. However, while these drugs provide potent analgesia, they also carry serious side effects, especially dose-dependent respiratory depression, which can lead to slowed or even stopped breathing—one of the most dangerous adverse reactions in clinical use. Furthermore, long-term or improper use can lead to tolerance, dependence, and addiction. Among the many fentanyl derivatives, carfentanil is one of the most pharmacologically active analogs, with a potency approximately 10,000 times that of morphine.

[0003] Currently, clinical treatment for the abuse and dependence caused by ultra-potent opioids like carfentanil still faces severe challenges. The mainstream treatment model primarily follows the traditional opioid dependence intervention pathway, focusing on drug replacement and adjunctive therapy. Commonly used drugs include: long-acting opioid receptor agonists, such as methadone, which alleviate cravings and withdrawal symptoms and reduce illicit drug use through long-acting, stable receptor agonism; partial receptor agonists, such as buprenorphine, which can reduce the risk of respiratory depression to some extent and are used for detoxification or maintenance therapy; and opioid receptor antagonists, such as naloxone, which can rapidly reverse respiratory depression caused by overdose. However, these traditional drugs have significant limitations: methadone and buprenorphine are still opioids, and long-term use may lead to addiction; they are also insufficient for controlling addiction in some patients addicted to potent derivatives; naloxone interferes with endogenous opioid peptides, causing side effects such as nausea and constipation, resulting in low patient compliance. Therefore, in the face of the unique challenges posed by highly potent opioids such as carfentanil, it is urgent to break out of the framework of traditional replacement therapy and explore more safe, efficient and mechanism-specific innovative strategies.

[0004] Vaccine technology, based on the immunological principle of antigen-antibody specific binding, has achieved great success in the field of infectious disease control. The "addiction vaccine" strategy, which induces the body to produce specific antibodies to neutralize addictive drugs and prevent them from entering the brain, offers a new approach to the treatment of substance dependence.

[0005] Current vaccine development strategies for opioid addiction prevention and treatment are mostly limited to simple physical mixing of hapten-carrier protein conjugates and adjuvants. These strategies suffer from insufficient antibody titers, severe carrier protein immunosuppression, low proportions of specific immunoglobulin (IgG) antibodies against drug molecules, and reliance on frequent booster immunizations to maintain immune memory, thus failing to provide lasting protection.

[0006] Therefore, there is an urgent need to construct a novel delivery system that can actively regulate the synergistic activation of innate and adaptive immunity by antigens and adjuvants, in order to achieve sustained and efficient immune intervention against opioid addiction. Summary of the Invention

[0007] The purpose of this section is to outline some aspects of the embodiments of the present invention and to briefly describe some preferred embodiments.

[0008] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0009] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a carfentanil molecular vaccine.

[0010] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a carfentanil molecular vaccine, wherein the general structural formula of the carfentanil molecular vaccine is shown in formula (I): (I) Wherein, A is a pattern recognition receptor agonist, B is an MHC class II restricted T-cell epitope peptide, C is a carfentanil hapten, m is 40-140, and n is 60-350.

[0011] In a preferred embodiment of the carfentanil molecular vaccine of the present invention, the carfentanil hapten is a carfentanil derivative, the general structural formula of which is shown in formula (II): (II) R1, R2, and R3 are H or connecting arms with active functional groups at the ends.

[0012] As a preferred embodiment of the carfentanil molecular vaccine of the present invention, wherein: the linking arm is selected from C2-C12 alkylene chains, polyethylene glycol chains or rigid linking groups containing benzene rings; The active functional group is selected from carboxyl, amino, mercapto, maleimide, azide, or alkynyl.

[0013] As a preferred embodiment of the carfentanil molecular vaccine of the present invention, wherein the pattern recognition receptor agonist is a Toll-like receptor 7 / 8 agonist.

[0014] As a preferred embodiment of the carfentanil molecular vaccine of the present invention, wherein the Toll-like receptor 7 / 8 agonist is IMDQ or a derivative thereof.

[0015] As a preferred embodiment of the carfentanil molecular vaccine of the present invention, wherein the MHC-II class molecular restriction T cell epitope peptide is a universal or carrier protein-derived helper T cell epitope. The universal helper T cell epitope is selected from one or more of the following: PADRE peptide, TT830-843 peptide derived from tetanus toxoid, OVA323-339 peptide derived from ovalbumin, or MVF peptide derived from measles virus fusion protein.

[0016] Another object of the present invention is to overcome the shortcomings of the prior art and provide a method for preparing a molecular vaccine, comprising, The core carrier polylactic acid-polyethyleneimine (PLA-PEI) block copolymer was covalently coupled sequentially or separately with pattern recognition receptor agonists, carfentanil haptens, and MHC class II restricted T-cell epitope peptides.

[0017] As a preferred embodiment of the method described in this invention, the covalent coupling reaction includes carbodiimide condensation reaction, click chemistry reaction, or Michael addition reaction.

[0018] Another object of the present invention is to overcome the shortcomings of the prior art and provide the use of a molecular vaccine in the preparation of a medicament for the prevention or treatment of carfentanil-related disorders.

[0019] As a preferred embodiment of the application described in this invention, the carfentanil-related disorders include carfentanil addiction, carfentanil overdose, or relapse after carfentanil use.

[0020] Beneficial effects of this invention: This invention relates to a method for preparing a PLA-PEI-based carfentanil molecular vaccine, which can produce high levels of specific antibodies against carfentanil; and to the application of the PLA-PEI-based carfentanil molecular vaccine in drugs for the prevention or treatment of carfentanil-related disorders. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein: Figure 1This is the synthetic route for the carfentanil hapten in Example 1 of the present invention.

[0022] Figure 2 This is the 1H NMR spectrum of the carfentanil hapten in Example 1 of the present invention.

[0023] Figure 3 This is the carbon spectrum of the carfentanil hapten in Example 1 of the present invention.

[0024] Figure 4 The carfentanil molecular vaccine MECV in Example 2 of this invention PLA-PEI The synthetic route.

[0025] Figure 5 This invention relates to the carfentanil molecular vaccine MECV. PLA-PEI The proton NMR spectrum.

[0026] Figure 6 This describes the immunization and blood collection procedures for the mice in Example 3, as well as the antibody titer determination results produced by the mice; wherein, Figure 6 'a' refers to the immunization and blood collection process. Figure 6 b is the structural formula of the ELISA coating antigen. Figure 6 cf represents the antibody titer measured in mice at days 21, 35, and 200. Figure 6 g represents the result of the assay for non-specific antibodies against the vaccine vector.

[0027] Figure 7 The carfentanil molecular vaccine in Example 4 can alleviate carfentanil-induced behavior, wherein... Figure 7 a is a schematic diagram of the hot plate experiment scheme. Figure 7 b. Hot plate assembly diagram Figure 7 c represents the effect of the carfentanil molecular vaccine on the analgesic effect induced by carfentanil. Figure 7 d is a schematic diagram of the conditional position preference (CPP) experimental design. Figure 7 e is a diagram of the CPP device. Figure 7 f represents the effect of the carfentanil molecular vaccine on carfentanil-induced CPP. Detailed Implementation

[0028] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0029] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from Shanghai Biotech Pharmaceutical Technology Co., Ltd. The thin-layer chromatography (TLC) equipment and silica gel used for analysis were from Qingdao Shuoyuan Silica Gel Technology Co., Ltd. The medium-pressure separation chromatography system was provided by Biotage. The silica gel column was purchased from Changzhou Sante Technology Co., Ltd. 1 H and13 C10 NMR spectra were recorded on a Bruker UltraShield™ spectrometer (300 MHz) in deuterated chloroform (CDCl3) or deuterated methanol (MeOD). Chemical shifts are expressed as δ in parts per million (ppm), with tetramethylsilane or solvent used as internal standards. 1 The coupling constant of H is denoted by J (Hz). Splitting modes in the spectra are represented as follows: singlet (s), doublet (d), triplet (t), quartet (q), doublet (dd), and overlapping signals are denoted as multiplets (m). Low-resolution electrospray (ES) mass spectra were recorded on a Waters QDa mass spectrometer, operating in both positive and negative ion modes.

[0030] Animal housing: Male C57 mice aged 6-8 weeks and weighing 20-28 g were housed in a temperature-controlled environment (21-24°C) following a 12-hour day-night cycle, with lights turned on at 08:00 AM. Mice had free access to standard rodent food and water and were allowed at least one week to acclimatize to the environment before any experimental procedures were performed.

[0031] ELISA assay (antibody titer determination): Equal doses were administered subcutaneously via tail injection on days 0, 14, and 28. The injection dose was 5-50 μg / animal (calculated as hapten). 100 µL of whole blood was collected from the retroorbital vein on days 21 and 35. The whole blood samples were incubated at 37°C for 1 hour, followed by centrifugation at 3500 rpm for 10 minutes to separate the serum. The serum was stored at -80°C until analysis. 96-well microplates (Costar 3690) were coated with PBS solution of BSA-carfentanil hapten conjugate (100 μL per well, concentration 0.5 μg / mL) and incubated overnight at 4°C. The plates were washed three times with PBST buffer (PBS containing 0.05% Tween-80). 100 µL of 5% skim milk solution was added to each well and the plates were blocked at room temperature for 2 hours, followed by three washes with PBST. Mouse serum was serially diluted with 5% skim milk in PBS solution for a total of 10 gradients, starting at a 1:256 ratio. 100 µL of each solution was added to a microplate and incubated at room temperature for 2 hours, followed by three washes with PBST. Then, 100 µL of HRP-conjugated affinipuregoat anti-mouse IgG (H+L) (Proteintech) (diluted 1:5000 in 5% skim milk in PBS solution) was added, and the plate was incubated at room temperature for 2 hours, followed by three washes with PBST. 100 µL of 3,3',5,5'-tetramethylbenzidine (TMB) reagent (Beyotime) was added to each well, and the plate was incubated at room temperature for 15 minutes. Finally, 100 µL of 2 M H2SO4 solution was added to each well to terminate the colorimetric reaction, and the absorbance at 450 nm was measured using a SYNERGY H1 microplate reader (BioTek Instruments, Carlsbad, CA, USA).

[0032] Hot plate test: Equal doses of the drug were administered subcutaneously via the tail on days 0, 14, and 28. The injection dose, calculated as hapten, was 5-50 μg / mouse. The hot plate test was performed 9 days after the third immunization. The hot plate apparatus consisted of a heated metal surface and a covered transparent box (Jinan Yiyan Technology Development Co., Ltd.). The temperature was set at 55.0 ± 0.1℃, and mice were placed individually on the heated surface. Animals would attempt to escape the noxious stimulus by lifting or licking their hind paws, jumping, etc. The latency period from contact with the hot plate to the emergence of escape behavior (i.e., reaction time) was defined as the pain threshold and recorded by a manual timer. The recording cutoff time was 45 seconds. If a mouse did not respond by jumping or licking its paw within 45 seconds, it was removed from the apparatus to avoid tissue damage.

[0033] Day 1: Place the mice in the hot plate device for adaptation training. Each mouse will be adapted for 5 minutes each time, for a total of 2 times, with an interval of 1 hour.

[0034] Day 2: Observe and record the pain threshold of each mouse. Measure each mouse twice, with a 1-hour interval. The average of the two test results is the baseline pain threshold.

[0035] On day 3: The PBS group was injected with physiological saline (5 mL / kg), while the other control group mice were injected intraperitoneally with carfentanil (10 μg / kg). After injection, the pain threshold of each mouse was recorded at 15, 30, 45 and 60 minutes, with a cutoff time of 60 seconds to avoid tissue damage.

[0036] The data is expressed as %MPE = (post-drug latency - baseline latency) / (cutoff time - baseline latency) × 100%.

[0037] Conditioned position preference (CPP) test: Mice were administered equal doses via subcutaneous tail injection on days 0, 14, and 28. The injection dose was 5–50 μg / mouse, calculated as hapten. CPP was assessed 10 days after the third immunization using an unbiased protocol in a three-compartment PVC device. The device consisted of two large chambers (15 cm × 15 cm × 40 cm) and a smaller central chamber (5 cm × 5 cm × 40 cm); the large chambers had different floor textures (stripes or grids) and wall colors (black or white) to provide different environmental cues. Mouse behavior was recorded by an overhead camera positioned 2 meters above the device and analyzed using EthoVisionXT.

[0038] Days 1-2: Allow the mice to acclimatize to the device for 15 minutes each day.

[0039] Day 3: Allow each mouse to freely explore all compartments for 15 minutes to determine its baseline preference.

[0040] Days 4-11: Mice were immediately subjected to 30 minutes of conditioning training after treatment, as follows: The PBS group was injected with saline daily, while the other control groups were injected with carfentanil (2.0 μg / kg) intraperitoneally on the drug day and with saline injected on the alternate days.

[0041] Day 12: Mice were allowed free access to all compartments for 15 minutes in a drug-free state. The time (in seconds) each mouse spent in each compartment was recorded. The CPP score was calculated by subtracting the time spent in the saline-paired compartment from the time spent in the drug-paired compartment.

[0042] Example 1 The synthesis of carfentanil hapten, the synthesis method is as follows: Figure 1 As shown, the specific steps are as follows: (1) Synthesis of compound 1.

[0043] 4-Piperidinone hydrochloride (2.5 g, 18.4 mmol) was dissolved in dichloromethane (45 mL), and di-tert-butyl dicarbonate (4.7 mL, 20.3 mmol) was added. Triethylamine (6.2 mL, 44.3 mmol) was then slowly added. The mixture was stirred at room temperature for 5 hours. After the reaction was complete, the solution was quenched with saturated sodium bicarbonate solution. The aqueous phase was extracted three times with dichloromethane. The combined organic phases were washed successively with brine, dried over anhydrous sodium sulfate, and filtered. The organic phase was concentrated by rotary evaporation and then separated by silica gel column chromatography to give compound 1 (3.0 g, 82% yield) as a white solid. Mp = 73.2–74.2 °C. 1 H NMR (300 MHz, CDCl3) δ 3.68 (t, J =6.2 Hz, 4H), 2.40 (t, J = 6.2 Hz, 4H), 1.45 (s, 9H). 13 C NMR (75 MHz, CDCl3) δ207.95, 154.53, 80.51, 41.22, 28.41. MS (ESI) m / z calcd for C 10 H 18 NO3 + [M+H] + 200.13, found 200.31. (2) Synthesis of compound 2.

[0044] Aniline (6.7 mL, 73.1 mmol) was dissolved in tetrahydrofuran (150 mL). Sodium hydroxide (1.7 g, 41.4 mmol), chloroform (3.3 mL, 41.4 mmol), and compound 1 (4.8 g, 24.4 mmol) were added sequentially in a 0°C reaction bath. The mixture was then stirred at room temperature for 6 hours. After the reaction was complete, the resulting suspension was filtered, the filter cake was dissolved in water, and the aqueous phase was extracted three times with diethyl ether.

[0045] The pH was then adjusted to 3 by adding 3M hydrochloric acid to the aqueous phase. The aqueous phase was extracted three times with ethyl acetate. The combined organic phases were washed successively with brine, dried over anhydrous sodium sulfate, and filtered. The organic phase was concentrated by rotary evaporation and then separated by silica gel column chromatography to give compound 2 (2.3 g, yield 29%) as a yellow solid. Mp = 127.2–129.0 °C. 1 H NMR (300 MHz, CDCl3) δ 7.15(t, J= 7.8 Hz, 2H), 6.97 – 6.47 (m, 5H), 3.91 – 3.52 (m, 2H), 3.52 – 2.99 (m,2H), 2.09 (dd, J = 18.9, 9.1 Hz, 2H), 1.94 (d, J = 13.6 Hz, 2H), 1.44 (s, 9H). 13 CNMR (75 MHz, CDCl3) δ 178.66, 154.98, 144.21, 129.32, 119.76, 116.31, 80.28,58.64, 28.49. MS (ESI) m / z calcd for C 17 H 25 N2O4 + [M+H] + 321.18, found 321.29. (3) Synthesis of compound 3. Propionic anhydride (14.7 mL, 115.0 mmol) was added to an ethyl acetate (300 mL) solution of compound 2 (18.4 g, 57.5 mmol), followed by triethylamine (24.0 mL, 172.5 mmol). The reaction was carried out at 40 °C for 9 hours. After the reaction was completed and cooled to room temperature, the mixture was diluted with water. The pH of the reaction mixture was adjusted to 3 by adding 3 M hydrochloric acid. The aqueous phase was extracted three times with ethyl acetate. The organic phases were combined, washed successively with brine, dried over anhydrous sodium sulfate, and filtered. The organic phase was concentrated by rotary evaporation and separated by silica gel column chromatography to give compound 3 (9.0 g, yield 42%) as a yellow solid. Mp = 166.0-168.0 °C. 1 H NMR (300 MHz, CDCl3) δ 8.42 (s, 1H), 7.69–7.10 (m, 5H), 3.74 (dd, J = 13.9,6.9 Hz, 2H), 3.25 (s, 2H), 2.30 (d, J = 13.2 Hz, 2H), 1.96 (q, J = 7.3 Hz, 2H),1.63 – 1.33 (m, 11H), 0.99 (t, J = 7.4 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 176.35,175.38, 154.85, 138.98, 130.43, 129.61, 129.06, 79.72, 62.97, 33.05, 29.20,28.45, 9.12. MS (ESI) m / z calcd for C 20 H 28 N2O5Na + [M+Na] + 399.19, found 399.33. (4) Synthesis of compound 4. Potassium carbonate (3.3 g, 23.8 mmol) was added to a DMF (50 mL) solution of compound 3 (4.5 g, 11.9 mmol), followed by potassium iodide (198 mg, 1.2 mmol) and tert-butyl 4-bromobutyrate (3.1 mL, 17.8 mmol). The reaction was carried out at 40 °C for 5 hours. After the reaction was completed, the solution was cooled to room temperature and diluted with water. The aqueous phase was extracted three times with ethyl acetate. The organic phases were combined, washed successively with brine, dried over anhydrous sodium sulfate, and filtered. The organic phase was concentrated by rotary evaporation and separated by silica gel column chromatography to give compound 4 (5.1 g, 83% yield), which was a yellow oil. 1 H NMR (300 MHz, CDCl3) δ 7.56 –7.35 (m, 3H), 7.27 (dd, J = 6.4, 3.7 Hz, 2H), 4.22 (t, J = 6.5 Hz, 2H), 3.74 (s,2H), 3.18 (s, 2H), 2.33 (t, J = 7.5 Hz, 2H), 2.25 (d, J = 13.6 Hz, 2H), 2.11 –1.93 (m, 2H), 1.87 (q, J = 7.4 Hz, 2H), 1.46 (dd, J = 28.2, 9.3 Hz, 20H), 0.95(t, J = 7.4 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 174.07, 172.94, 172.26, 154.78,139.22, 130.51, 129.62, 128.98, 80.58, 79.68, 64.21, 62.86, 33.23, 32.01,29.08, 28.43, 28.16, 24.25, 9.20. MS (ESI) m / z calcd for C 28 H 42 N2O7Na + [M+Na] + 541.29, found 541.39. (5) Synthesis of compound 5. Compound 4 (2.1 g, 4.0 mmol) was dissolved in trifluoroacetic acid (10 mL) and reacted at 40 °C for 12 hours. After the reaction was completed, the mixture was cooled to room temperature, concentrated by rotary evaporation, and separated by silica gel column chromatography to obtain a yellow oily compound 5 (624 mg, 43%). 1 H NMR (300 MHz, MeOD) δ 7.79 – 7.17 (m, 5H), 4.27 (t, J =6.1 Hz, 2H), 3.37 - 3.30 (m, 4H), 2.50 - 2.41 (m, 4H), 2.24 - 1.62 (m, 6H), 0.95 (t, J = 7.4 Hz, 3H). 13 C NMR (75 MHz, MeOD) δ 176.50, 176.48, 173.26,139.47, 131.50, 131.08, 130.68, 66.01, 61.90, 42.20, 31.50, 30.95, 29.82,25.07, 9.46. MS (ESI) m / z calcd for C 19 H 17 N2O5 + [M+H] + 363.19, found 363.32. (6) Synthesis of compound 6 (carfentanil hapten) 2-Bromoethylbenzene (0.22 mL, 1.6 mmol) was added to a DMF (5 mL) solution of compound 5 (483 mg, 1.3 mmol), followed by potassium iodide (45 mg, 0.3 mmol) and sodium bicarbonate (134 mg, 1.6 mmol). The reaction was carried out at 50 °C for 5 hours. After the reaction was completed and cooled to room temperature, the solution was diluted with water. The pH of the aqueous phase was adjusted to 3 by adding 3M hydrochloric acid. The aqueous phase was extracted three times with ethyl acetate. The organic phases were combined, washed successively with brine, dried over anhydrous sodium sulfate, and filtered. The organic phase was concentrated by rotary evaporation and then separated by silica gel column chromatography to give a yellow oily compound 6 (447 mg, 72% yield). 1 H NMR (300 MHz, MeOD) δ7.57 – 7.06 (m, 10H), 4.18 (t, J = 6.2 Hz, 2H), 3.43 (d, J = 12.7 Hz, 2H), 3.28 –3.13 (m, 4H), 2.99 – 2.83 (m, 2H), 2.44 (d, J = 14.8 Hz, 2H), 2.34 (t, J = 7.2Hz, 2H), 2.01 – 1.78 (m, 6H), 0.86 (t, J = 7.4 Hz, 3H). 13 C NMR (75 MHz, MeOD) δ176.84, 176.58, 173.18, 139.46, 137.53, 131.53, 131.13, 130.72, 129.94,129.81, 128.24, 66.13, 61.63, 58.76, 50.76, 31.78, 31.63, 31.37, 29.83,25.18, 9.48. MS (ESI) m / z calcd for C 27 H 35 N2O5 + [M+H] + 467.25, found 467.38. See the 1H NMR spectrum of the carfentanil hapten. Figure 2 See carbon spectrum Figure 3 .

[0046] Example 2 Carfentanil Molecular Vaccine (MECV) PLA-PEI Synthesis: A sequential covalent coupling strategy was employed to integrate the three functional components into the vector in sequence. The synthesis method is as follows: Figure 4 As shown, the specific steps are as follows: (1) Synthesis of PLA-PEI: Lactide was recrystallized three times with ethyl acetate and dried under vacuum at 40°C to constant weight; under nitrogen protection, 10 g of dried lactide and 10 mL of ultra-dry toluene (dried by calcium hydride and then distilled) were added to the reaction tube and stirred to dissolve. Stannous octoate (1 / 300 of the molar amount of lactide) and n-butanol (1 / 300 of the molar amount of lactide) were added in sequence; the reaction tube was sealed and stirred in an oil bath at 80°C for 24 hours; after the reaction, it was cooled to room temperature, and the reaction solution was dropped into excess ice-cold ethanol to precipitate. The precipitate was collected by filtration and dried under vacuum at 40°C to constant weight to obtain hydroxyl-terminated PLA oligomer (molecular weight 5000). PLA (1.0 mmol) was then dissolved in anhydrous DMSO (50 mL), followed by the addition of N'N-carbonyldiimidazole (CDI, 1.2 mmol). After stirring at room temperature for 12 hours, PEI (molecular weight 10000, 1.0 mmol) dissolved in anhydrous DMSO (50 mL) was added. Purification was performed by dialysis, starting with DMSO and gradually increasing the proportion of water until the system was completely replaced with an aqueous system. After freeze-drying, a white, fluffy solid product was obtained. 1 H NMR (300 MHz, D2O) δ 3.00 - 2.12 (m, 924H). 1 H NMR (300 MHz, D2O) δ 4.27 - 4.10 (m, 62H), 3.48 - 2.20 (m, 924H), 1.42 - 1.20 (m, 189H). (2) Synthesis of PLA-PEI-IMDQ conjugate: 1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-C]quinoline-4-amine (IMDQ, 0.06 mmol) was dissolved in DMSO, and CDI (0.09 mmol) was added and stirred at room temperature for 12 hours. Subsequently, a DMSO solution of PLA-PEI (molecular weight 15000, 0.02 mmol) was added to the activated IMDQ, and stirring was continued at room temperature for 72 hours. The purification process employs a stepwise solvent replacement method using dialysis: starting with DMSO, the solvent is gradually replaced by dialysis, and the proportion of water is gradually increased from DMSO until the system is completely replaced with an aqueous system. After lyophilization, a white, fluffy solid product is obtained. 1H NMR(500 MHz, D2O) δ 7.84 - 6.59 (m, 8H), 4.22 (d, J = 5.1 Hz, 77H), 3.80-1.86(m, 1266H), 1.54 - 1.07 (m, 251H), 0.83 (s, 3H). According to NMR calculations, the number of IMDQ couplings is 0.9.

[0047] (3) Synthesis of PLA-PEI-IMDQ-hapten-PDP conjugate: Carfentanil hapten (compound 6, 0.2 mmol) and 3-(2-pyridinedithio)propionic acid (PDP, 0.13 mmol) were dissolved in DMSO, and EDC (1.5 mmol) and NHS (0.5 mmol) were added. The mixture was stirred at room temperature for 6 hours. Subsequently, PLA-PEI-IMDQ conjugate (0.013 mmol) dissolved in DMSO was added to the activated carfentanil hapten and PDP system, and stirring was continued at room temperature for 72 hours. Purification was performed by dialysis, starting with DMSO and gradually increasing the proportion of water until the system was completely replaced with an aqueous system. After freeze-drying, a white, fluffy solid product was obtained. 1 H NMR (500 MHz, D2O) δ 8.39 (s, 1H), 8.17 (s, 1H),7.79 (s, 1H), 7.60 (s, 1H), 7.59 - 7.01 (m, 27H), 4.20 (d, J = 5.5 Hz, 28H),4.00 -2.60 (m, 322H), 2.53 - 2.18 (m, 19H), 2.16 - 1.67 (m, 20H), 1.56 - 1.03(m, 61H), 0.96 - 0.50 (m, 3H). According to NMR calculations, the number of hapten conjugates is 7.7 and the number of PDP conjugates is 3.5.

[0048] (4) PLA-PEI-IMDQ-hapten-PDP-MHC II peptide conjugate, i.e., carfentanil molecular vaccine MECV PLA-PEI The synthesis of.

[0049] PLA-PEI-IMDQ-hapten-PDP conjugate (0.01 mmol) was dissolved in DMSO and then added to MHC class II molecule-binding peptide (P240909-XT1197207, Jier Biochemical (Shanghai) Co., Ltd., 0.02 mmol). The mixture was stirred and reacted at room temperature for 72 hours. Purification was performed by dialysis, starting with DMSO and gradually increasing the proportion of water until the system was completely replaced with an aqueous system. After freeze-drying, a white, fluffy solid product was obtained.

[0050] Due to the complexity of the molecular structure, the number of MHC-binding peptides coupled was not calculated. Successful coupling at this stage can be achieved through... 1 The determination is made by the corresponding decrease in the intensity of the characteristic peak of PDP in H-NMR.

[0051] Carfentanil Molecular Vaccine (MECV) PLA-PEI See the proton NMR spectrum. Figure 5 .

[0052] (5) To verify the rationality of the design, a carfentanil molecular vaccine MECV based on PEI was also prepared. PEI .

[0053] Its conjugation process is the same as that of the PLA-PEI-based carfentanil molecular vaccine, except that the core vector is replaced by PEI instead of PLA-PEI.

[0054] PEI-IMDQ coupling: 1 ¹H NMR (500 MHz, D₂O) δ 7.82 - 6.33 (m, 8H), 4.21 (s, 2H), 3.20-1.80 (m, 845H), 1.58 (s, 2H), 1.20 (s, 2H), 0.85 (s, 3H). PEI-IMDQ-Hapten-PDP conjugate: 1 ¹H NMR (500 MHz, D₂O) δ 8.48 (s, 1H), 8.29 (s, 1H), 7.87 (s, 1H), 7.65 (s, 1H), 7.60 - 6.97 (m, 29H), 4.17 (s, 9H), 4.06 - 2.62 (m, 272H), 2.56 - 2.20 (m, 24H), 2.14 - 1.68 (m, 24H), 0.79 (s, 9H). Based on NMR calculations, MECV... PEI The numbers of IMDQ, hapten, and PDP conjugates were 1.1, 9.9, and 3.8, respectively.

[0055] (6) In order to compare with existing protein vaccines, the present invention prepared a vaccine OVA-Vax based on ovalbumin OVA as a carrier, and the preparation steps are as follows: Carfentanil hapten (0.1 mmol, 46.6 mg) was dissolved in DMSO (1 mL) containing 10% water, and then EDC (0.45 mmol) and NHS (0.45 mmol) were added. The mixture was stirred at room temperature for 4.5 hours. Subsequently, OVA solution (10 mg / mL, pH 7.4, 0.0005 mmol, 225 mg) was added to the activated hapten solution. The solution was stirred at 4 °C for 24 hours, and then purified five times by dialysis with 1×PBS (pH 7.4) (4 hours each time, 4 °C). After lyophilization, the resulting white fluffy solid was redissolved in PBS (pH 7.4), and the protein concentration was determined by the BCA method. To quantify the hapten density of OVA-Vax, the sample was analyzed by MALDI-TOF mass spectrometry and compared with uncoupled proteins. The results showed that each OVA molecule was coupled with an average of 47 haptens.

[0056] Example 3 MECV PLA-PEI Assay of antibody titers produced in mice Twenty mice were randomly divided into three groups: PBS group, OVA-Vax group, and MECV group. PEI Groups and MECV PLA-PEI Group (where OVA-Vax ovalbumin conjugated with carfentanil hapten was a positive control).

[0057] On days 0, 14, and 28, mice were administered an equal amount of carfentanil hapten subcutaneously via the tail. On days 21 and 35, 100 µL of whole blood was collected from the retroorbital vein. After serum separation, ELISA was performed. Figure 6 'a' refers to the immunization and blood collection process. Figure 6 b is the structural formula of the ELISA coating antigen.

[0058] The results showed that all experimental groups produced specific antibodies against the hapten, and antibody levels continued to rise with increasing number of immunizations. MECV PLA-PEI The midpoint titer induced by the vaccine was significantly higher than that induced by MECV. PEI The presence of OVA-Vax vaccines demonstrates their ability to more effectively elicit a potent humoral immune response against the target antigen. Furthermore, antibodies persist long after immunization, remaining detectable as late as 200 days post-primary immunization, suggesting MECV. PLA-PEI It may promote the sustained formation of immune memory. Figure 6 cf).

[0059] In addition, MECV PLA-PEI and MECV PEI Neither induced antibodies against the vector, while OVA-Vax produced an antibody response against the OVA vector itself. Figure 6 g).

[0060] In summary, MECV PLA-PEI Not only better than MECV PEI OVA-Vax can generate stronger and more durable hapten-specific antibody responses and can effectively avoid the generation of non-specific anti-vector immunity, fully demonstrating its potential as a highly efficient targeted vaccine delivery vector.

[0061] Example 4 MECV PLA-PEI It can reduce carfentanil-induced analgesia and addictive behavior: To evaluate MECV PLA-PEI The neutralizing capacity of vaccine-induced carfentanil-specific antibodies was investigated, and the blocking effect of immunized mice on the pharmacological effects of carfentanil was examined, with a focus on its analgesic effect and addiction potential.

[0062] Twenty mice were randomly divided into three groups: PBS group, carfentanil administration group (CAF group), and MECV group. PEI Groups and MECV PLA-PEI Group A received equal doses of carfentanil hapten (20 μg per mouse) via subcutaneous administration to the tail on days 0, 14, and 28. A hot plate test was performed 9 days after the third immunization. Figure 7 a is a schematic diagram of the hot plate experiment scheme. Figure 7 b. Hot plate assembly diagram Figure 7 d is a schematic diagram of the conditional position preference (CPP) experimental design. Figure 7 e is a diagram of the CPP device.

[0063] The results showed that mice in the carfentanil-treated group experienced a significantly prolonged latency period after administration of carfentanil (10 μg / kg), indicating that carfentanil has a significant analgesic effect. MECV PEI The analgesic effect in mice was not significantly different from that in the control group 15 minutes after administration, but the carfentanil-induced analgesia was significantly weakened at 30, 45 and 60 minutes. MECV PLA-PEI The carfentanil-induced analgesia group showed a more significant inhibitory effect at all time points, with the inhibitory effect at 15 minutes being particularly strong compared to MECV. -PEI Group( Figure 7 c).

[0064] Meanwhile, another 20 mice were randomly divided into PBS group, CAF group, and MECV group. PEI Groups and MECV PLA-PEIIn this group, mice were administered carfentanil hapten subcutaneously via the tail on days 0, 14, and 28. Ten days after the third immunization, the CPP test was performed. Results showed that mice in the carfentanil-treated group (2 μg / kg) had significantly higher CPP scores. Meanwhile, MECV... PEI and MECV PLA-PEI The expression of carfentanil-induced CPP in mice in the group was significantly reduced, indicating that the vaccine effectively attenuated its addictive effects. Figure 7 f).

[0065] In summary, MECV PEI and MECV PLA-PEI All of them can induce specific antibodies against carfentanil and inhibit its analgesic and addictive behaviors, among which MECV PLA-PEI It shows superior potential in suppressing early analgesic responses.

[0066] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A carfentanil molecular vaccine, characterized in that: The general structural formula of the carfentanil molecular vaccine is shown in formula (Ⅰ): （Ⅰ） Among them, A is a pattern recognition receptor agonist, B is an MHC class II restricted T cell epitope peptide, and C is a carfentanil hapten. m is 40-140, and n is 60-350.

2. The carfentanil molecular vaccine as described in claim 1, characterized in that: The carfentanil hapten is a carfentanil derivative, and its general structural formula is shown in formula (II): （Ⅱ） R1, R2, and R3 are H or connecting arms with active functional groups at the ends.

3. The carfentanil molecular vaccine as described in claim 2, characterized in that: The connecting arm is selected from C2-C12 alkylene chains, polyethylene glycol chains, or rigid connecting groups containing benzene rings; The active functional group is selected from carboxyl, amino, mercapto, maleimide, azide, or alkynyl.

4. The carfentanil molecular vaccine as described in claim 1, characterized in that: The pattern recognition receptor agonist is a Toll-like receptor 7 / 8 agonist.

5. The carfentanil molecular vaccine as described in claim 4, characterized in that: The Toll-like receptor 7 / 8 agonist is IMDQ or a derivative thereof.

6. The carfentanil molecular vaccine as described in claim 1, characterized in that: The MHC-II class molecule-restricted T-cell epitope peptides are universal or carrier protein-derived helper T-cell epitopes; The universal helper T cell epitope is selected from one or more of the following: PADRE peptide, TT830-843 peptide derived from tetanus toxoid, OVA323-339 peptide derived from ovalbumin, or MVF peptide derived from measles virus fusion protein.

7. A method for preparing the molecular vaccine according to any one of claims 1 to 6, characterized in that: include, The core carrier polylactic acid-polyethyleneimine (PLA-PEI) block copolymer was covalently coupled sequentially or separately with pattern recognition receptor agonists, carfentanil haptens, and MHC class II restricted T-cell epitope peptides.

8. The method as described in claim 7, characterized in that: The covalent coupling reaction includes carbodiimide condensation, click chemistry, or Michael addition.

9. Use of the molecular vaccine according to any one of claims 1 to 6 in the preparation of a medicament for the prevention or treatment of carfentanil-related disorders.

10. The application as described in claim 9, characterized in that: The carfentanil-related disorders include carfentanil addiction, carfentanil overdose, or relapse after carfentanil use.

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